Virology 476 (2015) 233–239

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Adaptive amino acid substitutions enhance the virulence of a reassortant H7N1 avian influenza virus isolated from wild waterfowl in mice Zhijun Yu a,b,1, Weiyang Sun b,1, Xue Li b,e,1, Qiang Chen c,d, Hongliang Chai c, Xiaolong Gao b, Jiao Guo b, Kun Zhang b, Tiecheng Wang b, Na Feng b, Xuexing Zheng b, Hualei Wang b, Yongkun Zhao b, Chuan Qin a, Geng Huang b, Songtao Yang b, Yuping Hua c, Xuemei Zhang e,n, Yuwei Gao b,f,nn, Xianzhu Xia a,b,f,nnn a

Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, People’s Republic of China Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, Changchun 130122, People’s Republic of China c College of Wildlife Resources, Northeast Forestry University, Harbin 150040, People’s Republic of China d Liaoning Medical University, Jinzhou 121001, People’s Republic of China e Changchun Institute of Biological Products, Changchun 130122, People’s Republic of China f Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, People’s Republic of China b

art ic l e i nf o

a b s t r a c t

Article history: Received 22 September 2014 Returned to author for revisions 20 November 2014 Accepted 24 November 2014

H7 avian influenza viruses (AIVs) have caused a number of human infections, highlighting the pandemic potential of them. However, the factors that promote their replication in mammals remain poorly understood. Here, we generated mouse-adapted variants of a reassortant H7N1 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the variants were reduced 410,000-fold compared to the parental virus. Adapted variants displayed enhanced replication kinetics in vitro and vivo, and were capable of replicating in multiple organs. Analysis of the variant virus genomes revealed amino acid changes in the PB2 (E627K), HA (H3 numbering; E114K, G205E, and G218E), and NA (S350N) proteins. Notably, some amino acid changes have been identified in natural H7 isolates. Our results implicate a number of amino acid substitutions that collectively enhance the ability of a wild bird-origin H7N1 AIV to replicate and cause severe disease in mice. & 2014 Elsevier Inc. All rights reserved.

Keywords: Wild waterfowl Avian influenza virus H7N1 Mice Adaptation

Introduction Wild aquatic birds are the natural reservoir of avian influenza viruses (AIVs) (Webster et al., 1992). Although AIVs usually replicate efficiently in their natural avian hosts, there are reports that some AIVs can also infect dogs, cats, plateau pikas, rhesus macaques, and occasionally humans (Cheng et al., 2014b; Shinya et al., 2012; Songserm et al., 2006; Yu et al., 2014; Zhang et al., 2013a, n

Corresponding author. Tel./fax: þ 86 431 85077196. Corresponding author at: The Military Veterinary Institute, Academy of Military Medical Science of PLA 666, Liuyingxi st., Changchun 130122, People’s Republic of China. Tel./fax: þ86 431 8698 5516. nnn Corresponding author at: The Military Veterinary Institute, Academy of Military Medical Science of PLA 666, Liuyingxi st., Changchun 130122, People’s Republic of China. Tel./fax: þ86 431 8698 5516. E-mail addresses: [email protected] (X. Zhang), [email protected] (Y. Gao), [email protected] (X. Xia). 1 These authors contributed equally to the results of this study. nn

http://dx.doi.org/10.1016/j.virol.2014.11.031 0042-6822/& 2014 Elsevier Inc. All rights reserved.

2013b). For example, human infections with a novel H7N9 AIV were first reported in China in 2013, and have been a growing public health concern (Gao et al., 2013). Notably, some AIVs have the capacity to transmit between ferrets and guinea pigs, further suggesting their pandemic potential (Belser et al., 2013; Gao et al., 2009; Zhang et al., 2013c). H7N1 viruses circulate worldwide in areas including North America (Panigrahy et al., 1995) and Europe (Brown, 2010; Gonzales et al., 2011), as well as southern China (Peng et al., 2014), Australia (Bulach et al., 2010), and New Zealand (Bulach et al., 2010). Zoonotic transmission of H7 influenza viruses to humans has been suggested by reports of significantly elevated H7-specific antibody titers among poultry workers in Italy (Di Trani et al., 2012), and documented H7 AIV infection of humans in China in 2013 (Gao et al., 2013). A previous report found that an H7N1 AIV with no history of human infection could become capable of airborne transmission among ferrets following adaptive changes introduced by serial passage (Sutton et al., 2014). Taken

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together, sporadic human infections with H7 AIVs, the ability of adapted H7N1 AIVs to transmit between mammals, and a lack of pre-existing immunity to H7N1 AIVs in humans suggest that H7N1 AIVs may pose a pandemic threat. In addition, the occurrence of the novel human H7N9 AIVs, which are low pathogenic avian influenza (LPAI) viruses, in 2013 suggested that the risk of H7 LPAI viruses infection in humans. Therefore, the molecular features involved in the mammalian adaptation of H7N1 LPAI viruses should be further studied. Mice provide an excellent animal model in which to study of the mammalian adaptation of avian influenza viruses (Belser et al., 2013; Belser and Tumpey, 2013; Cheng et al., 2014a; Gabriel et al., 2005; Hatta et al., 2001; Li et al., 2014, 2005; Song et al., 2009; Tumpey et al., 2002). To identify changes that are associated with the adaptation of H7N1 AIV to mammals, we serially passaged a wild waterfowl-origin H7N1 virus in mice. We hypothesized that serial passage would allow us to identify molecular determinants that are important for the adaptation of H7N1 AIVs to mammalian hosts through the purifying selection of viral variants with increased fitness. After sequential passage of an H7N1 virus in mice, we obtained two viruses that displayed enhanced replication kinetics and increased virulence in vitro and in vivo. Genomic analysis identified multiple amino acid substitutions in the adapted viral variants. These results identify a number of amino acid substitutions that collectively confer increased virulence, expanded viral tropism, and enhanced replicative capacity to a wild bird-origin H7N1 AIV in a mammalian model.

Results

body weight loss. A group of mock-infected mice were included as a control. Mice that lost 425% of their initial body weight were considered moribund and were euthanized. At an infecting dose of 106 EID50, 60% mice inoculated with WT 29Y survived, and showed modest body weight loss beginning approximately 3 days postinfection (Fig. 1A and B). In contrast, mice infected with 106 EID50 of the mouse-adapted MA-P1P5 or MA-P3P5 variants rapidly lost weight and succumbed to infection by day 3 post-infection (Fig. 1A and B). We determined that the WT 29Y virus MLD50 was 6.25 log10 EID50, and the MLD50 of the MA-P1P5 and MA-P3P5 viruses were 2.25 log10 EID50 and 1.75 log10 EID50, respectively (Table 2). Therefore, serial passage of WT 29Y resulted in substantially increased virulence in mice.

The mouse-adapted viruses replicate more efficiently in MDCK cells and A549 cells We next evaluated the replicative ability of the WT 29Y and mouse-adapted H7N1 strains in MDCK cells (Fig. 2A) and A549 cells (Fig. 2B). In vitro growth kinetics revealed that the MA-P1P5 and MA-P3P5 viruses grew faster and achieved higher titers than WT 29Y (Fig. 2A and B). MA-P3P5 grew to the highest titer and yielded approximately 14-fold higher viral titers than WT 29Y in MDCK cells by 48 h post-inoculation (Fig. 2A). MA-P3P5 grew to the highest titer and yielded approximately 10-fold higher viral titers than WT 29Y in A549 cells by 48 h post-inoculation (Fig. 2B). These data show that the enhanced virulence of the mouseadapted H7N1 viruses correlates with increased viral replication in vitro.

WT H7N1 29Y virus is a reassortant virus The genomic segments of the WT H7N1 29Y virus were sequenced and compared to the genomic sequences of other AIVs available in GenBank. Virus isolates possessing the highest percent nucleotide identity to each viral gene segment of the 29Y virus were identified and revealed that the WT H7N1 29Y virus is likely a reassortant viruses containing genes from H7N7, H7N1, H6N1, and H10N9 subtypes (Table 1). Sequence analysis show that the WT H7N1 29Y virus lacks a polybasic cleavage site in the hemagglutinin, suggesting that it is a LPAI virus for poultry. Serial passage generated H7N1 variants that are highly pathogenic in mice We compared the pathogenicity of the WT H7N1 29Y virus with two mouse-adapted variants (named MA-P1P5 or MA-P3P5) that were produced by independent serial passages of 29Y in mice. Mice were infected intranasally with 106 EID50 WT 29Y or the mouse-adapted variant viruses and were followed for 14 days for

The mouse-adapted H7N1 viruses replicate to higher titers in respiratory tissues and digestive tracts and acquired the ability to replicate in multiple organ systems in mice To determine if the mouse-adapted H7N1 strains also replicate more efficiently than the wild type virus in vivo, we inoculated mice with 104 EID50 of each virus and analyzed virus titers in the lungs on day 3 and 5 post-infection (Fig. 2C). Both mouse-adapted viruses replicated to significantly higher titers than WT 29Y in the lungs at day 3 post-infection, but replicated to levels comparable to WT 29Y by day 5 post-infection (Fig. 2C). The titers of WT 29Y were 6.9 7 0.4 log10 EID50/g on day 3 post-infection, and 7.7 70.4 log10 EID50/g on day 5 post-infection, whereas the titers of MAP1P5 and MA-P3P5 were 8.5 70.1 log10 EID50/g and 8.6 7 0.2 log10 EID50/g, respectively, on day 3 post-infection, and 7.4 70.2 log10 EID50/g and 7.970.1 log10 EID50/g on day 5 post-infection (Fig. 2C). These data show that the enhanced virulence of the mouseadapted H7N1 viruses correlates with increased viral replication in the lungs of mice by 3 days post-infection.

Table 1 Identification of viruses harboring gene segments with highest sequence identity to each segment of A/Baer’s Pochard/HuNan/414/2010 (H7N1) using sequence data available in GenBank. Genea

Virus with the highest percentage of nucleotide identity

GenBank accession no.

% Identity

PB2 PB1 PA HA NP NA M NS

A/mallard/Korea/NHG187/2008(H7N7) A/mallard/Sweden/1985/2003(H7N7) A/wild bird/Korea/A330/2009(H7N7) A/ruddy shelduck/Mongolia/921C2/2009(H7N1) A/mallard/Korea/NHG187/2008(H7N7) A/duck/Eastern China/1/2008(H6N1) A/northern shoveler/Hong Kong/MPE2531/2008(H10N9) A/duck/Mongolia/867/2002(H7N1)

KC609821 CY183342 JN244164 KC904723 KC609905 JF965336 KF259266 AB473547

99 98 99 99 99 99 99 99

a PB2, basic polymerase 2; PB1, basic polymerase 1; PA, acidic polymerase; HA, hemagglutinin; NP, nucleoprotein; NA, neuraminidase; M, matrix; NS, nonstructural protein.

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Sequence analysis

Fig. 1. Increased virulence of mouse-adapted H7N1 virus in mice. Mice (n¼ 5) were inoculated intranasally with 50 μl containing 106 EID50 of mouse-adapted isolates (MA-P1P5 and MA-P3P5) or the parental wild-type H7N1 virus (WT) or mock inoculated (Mock; n ¼5), and animals that lost more than 25% of their pre-infection weight were euthanized. (A) Morbidity was examined by recording the body weights of inoculated mice daily, and it is represented as a percentage of the weight on the day of inoculation (day 0). The average of each group is shown. The proportion of surviving mice in each group is indicated. (B) Mouse mortality after inoculation with 50 μl containing 106 EID50 of mouse-adapted isolates (MA-P1P5 and MA-P3P5) or the parental wild-type H7N1 virus (WT) or diluent (Mock).

We sequenced the genomes of the MA-P1P5 and MA-P3P5 viruses to identify the adaptive mutations responsible for the increased virulence in mice (Table 3). While sequence analysis revealed a number of synonymous and nonsynonymous mutations in the mouse adapted viruses, we focused on mutations that encoded amino acid substitutions for subsequent analyses. The MA-P1P5 and MA-P3P5 viruses both had an E-K substitution at PB2 position 627, a E-K substitution at HA position 122 (H7 numbering; H3 numbering is 114), a G-E substitution at HA position 214 (H7 numbering; H3 numbering is 205), a G-E substitution at HA position 227 (H7 numbering; H3 numbering is 218), and a S-N substitution at NA position 350 (Table 3). The MA-P3P5 virus also had an I-T substitution at PB2 position 615 that was not present in the MA-P1P5 virus (Table 3). To investigate whether the amino acid changes observed in mouse-adapted H7N1 strains generated in this study have been identified in H7 isolates, we queried H7 sequences deposited in the GenBank database. We identified three isolates possessing a T residue at PB2 position 615, forty-five isolates possessing a K residue at PB2 position 627, five isolates possessing a K residue at HA position 122 (H7 numbering), thirteen isolates possessing an E residue at HA position 214 (H7 numbering), and nine isolates possessing an E residue at HA position 227 (H7 numbering) (Table 4), and found no evidence of H7 isolates possessing an N residue at NA position 350. It is worth noting that the E627K substitution in the PB2 protein is known to play important roles in the adaptation of avian H5N1 viruses to mammalian hosts, and is associated with increased replicative capacity and pathogenesis in humans and mice (Gabriel et al., 2013; Hatta et al., 2001). Our results suggest that PB2 E627K may also be an important adaptive change leading to enhanced H7N1 viral virulence in mammals.

Discussion Table 2 MLD50 of wild-type 29Y H7N1 virus and mouseadapted H7N1 variants in BALB/c mice. Virusa

MLD50 (log10 EID50)

WT 29Y MA-P1P5 MA-P3P5

6.25 2.25 1.75

a WT 29Y, A/Baer’s Pochard/HuNan/414/2010 (H7N1); MA-P1P5, mouse-adapted 29Y; MA-P3P5, mouse-adapted 29Y.

We then asked whether the increased virulence of the mouseadapted H7N1 strains was due to an expansion of the tropism of the adapted variants. Mice were inoculated intranasally with 106 EID50 WT 29Y, MA-P1P5, or MA-P3P5. On day 3 post-infection, the lungs, brains, intestines, livers, spleens, and kidneys were collected and the viral load in each tissue was determined (Fig. 2D). Virus was recovered from the lungs and intestines of mice inoculated with WT 29Y, but was not detected in any other organ tested (Fig. 2D). In contrast, virus from mice inoculated with either mouse-adapted H7N1 strains were recovered from the lungs and intestines at higher titers when compared to mice inoculated with WT 29Y and were also recovered from the brains, livers, spleens, and kidneys (Fig. 2D). These data demonstrate that the mouseadapted H7N1 strains replicated to higher titers in respiratory tissues and digestive tracts when compared to the WT 29Y virus, and acquired the ability to replicate to detectable levels in the brain, liver, spleen, and kidney.

Considering that the LPAI viruses in poultry also pose severe threat to human health, as observed in H7N9 viruses (Gao et al., 2013), the WT H7N1 29Y virus, which is a LPAI virus for poultry, is used for studying the pathogenesis of AIVs in this research. In this study, we used mice as a model system for studying the mammalian adaptation of a reassortant H7N1 avian influenza virus isolated from wild waterfowl and show that highly pathogenic variants can quickly emerge from a parental virus during limited serial passage. These adapted viral variants displayed expanded tissue tropism and increased replication kinetics in vitro and in vivo. Sequence analysis of adapted viruses identified multiple amino acid substitutions, including a lysine residue at PB2 position 627, that are likely involved in the adaptation of H7N1 avian influenza virus in mice. Serial passage of avian influenza viruses has been frequently used to identify adaptive changes that can occur when avian viruses replicate in mammalian hosts (Gabriel et al., 2013, 2005). It is becoming increasingly clear that adaptive mutations are required in order for avian influenza viruses to efficiently replicate in mammalian hosts (Bussey et al., 2011). The PB2-E627K substitution has been consistently found to contribute to the adaptation of H5N1 and H7N7 highly pathogenic avian influenza viruses in mammals (Gabriel et al., 2005; Hatta et al., 2001). It is still unclear how PB2 E627K mutation changes viral pathogenicity in mammals, although the difference in surface charge of the 627Eor 627K-containing domain of PB2 may affect PB2 interactions with other viral or cellular proteins (Kuzuhara et al., 2009). While the PB2 E627K substitution has been reported to confer increased virulence in mammals in the context of other avian influenza

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Fig. 2. Growth characteristics of mouse-adapted H7N1 viruses in vitro and vivo. (A) MDCK cells were inoculated at a multiplicity of infection of 0.01 TCID50/cell with the parental wild-type H7N1 virus (WT) or a mouse-adapted virus (MA-P1P5 or MA-P3P5). Supernatants were collected at the indicated time points and titrated in MDCK cells by TCID50. (B) A549 cells were inoculated at a multiplicity of infection of 0.01 TCID50/cell with the parental wild-type H7N1 virus (WT) or a mouse-adapted virus (MA-P1P5 or MA-P3P5). Supernatants were collected at the indicated time points and titrated in MDCK cells by TCID50. (C) Mice (n ¼3) were inoculated intranasally with 104 EID50 of the parental wild-type H7N1 virus (WT) or a mouse-adapted virus (MA-P1P5 or MA-P3P5). Viral loads in the lungs were determined at 3 and 5 days post-infection in eggs by EID50. Results are expressed as log10 EID50/g of tissue. The dotted line indicates the limit of detection. (D) Mice (n ¼3) were infected with 106 EID50 of the parental wild-type H7N1 virus (WT) or a mouse-adapted virus (MA-P1P5 or MA-P3P5). On day 3 post-infection, viral loads in the lungs, brains, intestines, livers, spleens, and kidneys were titrated in eggs by EID50. Results are expressed as log10 EID50/g of tissue. The dotted indicates the lower limit of detection of infectious virus. In each panel, the average of three replicates is shown with error bars indicating the standard deviation. n, 1p o 0.05, when comparing MA-P1P5 and MA-P3P5 with WT respectively, as determined by two-way ANOVA. nn, 11p o0.01, when comparing MA-P1P5 and MA-P3P5 with WT respectively, as determined by two-way ANOVA. nnn, 111p o0.001, when comparing MAP1P5 and MA-P3P5 with WT respectively, as determined by two-way ANOVA.

Table 3 Nucleotide and amino acid substitutions identified in mouse-adapted H7N1 avian influenza viruses. Segment Nucleotide position

Nucleotide mutation

Amino acid position

Amino acid substitution

Mouseadapted virus

PB2

1844 1879

T-C G-A

615 627

I-T E-K

1077 364

C-T G-A

359 122a (114b)

– E-K

MA-P3P5 MA-P1P5 MA-P3P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P1P5 MA-P3P5 MA-P3P5

PA HA

432

C-T

a

b

–

a

a

144 (136 )

c

641

G-A

214 (205 )

G-E

680

G-A

227a (218b)

G-E

NP

1200 1260

R-A C-T

400a (390b) 420

– –

NA

1049

G-A

350

S-N

1116

C-T

372

a b c

H7 numbering. H3 numbering. Synonymous mutation.

subtypes, it has not been experimentally investigated for H7N1 subtype avian influenza viruses. In this report, we provide data to suggest that the PB2-E627K contributes to the highly virulent

Table 4 Web search for the amino acid changes observed in mouse-adapted H7N1 strains generated in this study have been identified in H7 subtype isolates. Segment

Position

Amino acid

Frequency of substitution (no. of strains with the substitution/total no. of strains)

PB2

615 627 122 214 227 350

T K K E E N

3/1469 45/1469 5/1951 13/1951 9/1951 0/1571

HA

NA

phenotype of mouse-adapted H7N1 viral variants. Multiple studies have linked amino acid substitutions in the HA protein of influenza viruses with enhanced mouse infectivity and virulence (Uraki et al., 2013; Watanabe et al., 2013). In our study, three HA amino acid substitutions ((H3 numbering; E114K, G205E, and G218E) were identified in two independently generated mouse-adapted variant viruses, suggesting that these adaptive changes may contribute to the enhancement of virulence of H7N1 AIV in mice. Differences in protease sensitivity of HA is an important determinant for systemic spread of infection (Bottcher-Friebertshauser et al., 2014), suggesting that these HA mutations found in our study may contribute to the expanded tissue tropism of these adapted H7N1 variants. Interestingly, the HA-G218E was also identified in the mouse-adapted human H3N2 influenza virus which exhibited high virulence in a previous report (Narasaraju et al., 2009).

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Importantly, many of the amino acid changes identified in the mouse-adapted H7N1 viruses have been identified in H7N1 isolates collected from various avian species (Table 4). In conclusion, we identified multiple amino acid substitutions that collectively enhance H7N1 AIV virulence in a mouse model. Our findings showed that increased H7N1 virulence was associated with accelerated viral growth in mammalian cells and expanded tissue tropism in vivo. Additional studies will be required to study the impact of each of these adaptive changes on viral virulence in mice. Mouse passage experiments are a way to identify adaptive changes that contribute to viral pathogenicity in mice. However, these changes may not have similar effects in other mammalian species. Human seasonal viruses, for example, are already well adapted to mammals, but often mutate to become more pathogenic in mice after passage in these animals. Therefore, extension of these findings to other animal models, such as ferrets and macaques, will be needed to better understand the mechanisms of mammalian adaptation and interspecies transmission of H7N1 AIVs and assess the impact of these amino acid substitutions on H7N1 infection of humans.

Materials and methods Facilities Studies with H7N1 AIV and the variants were conducted in a biosecurity level 3 laboratory approved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences. All animal studies were approved by the Review Board of Military Veterinary Research Institute of the Academy of Military Medical Sciences. Cells and virus Madin–Darby canine kidney (MDCK) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM glutamine, 5% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 mg/ml streptomycin. A549 cells were cultured in DMEM supplemented with 2 mM glutamine, 10% FCS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The H7N1 virus A/Baer’s Pochard/ HuNan/414/2010 (H7N1) (abbreviated as 29Y), was isolated from a Baer’s Pochard in Hunan province, China. Stock viruses were grown in the allantoic cavities of 10-day-old chicken eggs for 48 h at 37 1C, and aliquots were stored at  80 1C until used. The GenBank accession numbers corresponding to each of the eight 29Y viral gene segments are JQ973643-JQ973650. Adaptation of H7N1 influenza virus in mice Two mouse-adapted variants of the 29Y virus were derived from two independent series of sequential lung-to-lung passages of virus in mice as described previously (Brown, 1990). Briefly, female 3–5-week-old BALB/c mice (Merial-Vital Laboratory Animal Technology Co., Ltd., Beijing, China) were inoculated intranasally with 50 μl of allantoic fluid containing the wild type (WT) 29Y H7N1 virus under light isoflurane anesthesia. Lungs were harvested and homogenized 48 h after infection. The disrupted lung tissue was centrifuged to remove debris and 50 μl of the supernatant was used to inoculate the next naïve mouse in the series. Virus was passaged mouse-to-mouse five times in two independent lines. After the fifth passage in each series of mice, viruses present in the final lung homogenates were cloned once by plaque purification in MDCK cells as described previously (Song et al., 2009). The plaque-purified viruses were independently amplified in the allantoic cavities of 10-day-old chicken eggs for 48 h at 37 1C

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to prepare virus stocks. Two plaque-purified mouse-adapted variants of the original 29Y virus were obtained for further characterization and named MA-P1P5 and MA-P3P5. Virus titration in eggs and MDCK cells Virus titers in virus stocks and homogenized organ samples were determined by end-point titration in eggs and/or MDCK cells. For end-point viral titration in eggs, ten-fold serial dilutions of each sample were inoculated into eggs. Forty-eight hours after inoculation, fluid from the allantoic cavity was collected and tested for the ability to agglutinate chicken erythrocytes as an indicator of virus replication. Infectious virus titers were reported as log10 EID50/ml or log10 EID50/g, and were calculated from 3 replicates by the method of Reed and Muench (1938). For end-point viral titration on MDCK cells, ten-fold serial dilutions of each sample were inoculated onto MDCK cell monolayers grown in a 96 well culture plate. One hour after inoculation, the monolayer was washed with phosphate-buffered saline (PBS), and cultured in 100 ml of DMEM supplemented with 100 IU/ml penicillin, and 100 mg/ml streptomycin, 2 mM glutamine, and 2 mg/ml TPCKtreated trypsin. Seventy-two hours after inoculation, supernatants of infected cell cultures were tested for agglutinating activity using chicken erythrocytes as an indicator of virus replication. Infectious virus titers were calculated from 3 replicates by the method of Reed and Muench (1938), and expressed as log10 TCID50 (50% tissue culture infectious dose)/ml. In vivo experiments The 50% mouse lethal dose (MLD50) of the parental 29Y virus and the two mouse-adapted isolates was measured using groups of three female 4–5-week-old BALB/c mice (Merial-Vital Laboratory Animal Technology Co., Ltd., Beijing, China). Mice were intranasally inoculated with 50 μl of 10-fold serial dilutions of each indicated influenza virus in PBS under isoflurane sedation, with doses ranging from 101 to 106 EID50. Survival and body weight changes were recorded daily for 14 days post-infection (dpi). Animals that showed signs of severe disease and weight loss 425% of their initial body weight were considered moribund and were humanely sacrificed. MLD50 values were calculated by the Reed and Muench (1938) method after a 14-day observation period and expressed as EID50. Mice were additionally intranasally inoculated with 104 EID50 of the indicated viruses to measure the replicative capacity of mouseadapted isolates as compared to the parental 29Y virus in the lungs. Three mice in each group were euthanized at 3 and 5 dpi. The lungs were collected, homogenized, and tissue debris was removed by lowspeed centrifugation. Virus titers of each sample were determined by endpoint titration in eggs as described above, and virus titers of each strain were expressed as mean log10 EID50/g7standard deviation (SD). To evaluate the tropism and replication capacity of each virus in vivo, we euthanized three mice on 3 days after inoculation with 106 EID50 29Y or mouse-adapted isolates and harvested the lungs, brains, intestines, livers, spleens, and kidneys. Organs were homogenized in 1 ml of PBS and viral titers in each of the organs were determined by titration in chicken eggs. Titers were calculated by the Reed and Muench (1938) method and expressed as mean log10 EID50/g7SD. The limit of virus detection was 0.75 log10 EID50/g. For calculation of the mean, samples with a virus titer of o0.75 log10 EID50/g were assigned a value of 0. Analysis of replication kinetics in MDCK cells and A549 cells MDCK cells and A549 cells were infected with indicated influenza viruses at a multiplicity of infection (MOI) of 0.01

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TCID50/cell. After incubation, the cells were washed and overlaid with DMEM containing 2 mg/ml TPCK-treated trypsin. Supernatants were collected 12, 24, 36, 48, 60, and 72 h after infection and stored at  80 1C. Virus titer was determined by end-point titration in MDCK cells. Virus titers of each virus in different time point were expressed as mean log10 TCID50/ml 7 SD. RNA isolation, PCR amplification, and sequencing Viral RNA was isolated from the allantoic fluid of inoculated eggs using the RNeasy Mini kit (QIAGEN, Germantown, MD) according to the manufacturer’s protocol. Reverse transcription of viral RNA and subsequent PCR were performed using primers specific for each gene segment (sequences available upon request). PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Germantown, MD) according to the manufacturer’s protocol. Viral gene segments were sequenced by the Beijing Genomics Institute (Beijing, China). Sanger sequencing methodology was used to sequence the PCR-amplified viral gene segments. DNA sequences were analyzed and compared to the parental 29Y virus using the Lasergene sequence analysis software package (DNASTAR, Madison, WI).

Statistical analysis Data were analyzed by two-way analysis of variance using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). When a significant effect was observed, pairwise comparisons were performed using the Bonferroni post-hoc test.

Acknowledgments We thank Peter Wilker for editing the manuscript. This work was supported by the National High Technology Research and Development Program (No. 2012AA022006) and the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012ZX10004-502).

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